Letters in Applied Microbiologj 1992, 14,43-46

Increase of xanthan production by cloning xps genes into wild-type Xanthomonas campestris YI-HSIUNG T s F , N G ' ~ ~WEN-YEN *, T I N G ' ,H U E Y - C HCHOU', I BIH-YING YANG'? & C H I - C H E NC G H E NDepartment ~ of Botany', Institute of Molecular Biology2 and 3Department of Animal Science, National Chung Hsing University, Taichung 400, Taiwan J R N I 1 1 8 ; received 3 October 1991 and accepted 8 October 1991 T S E N GYI-HSIUNC,, , T I N G WEN-YEN, , C H O U , H U E Y - C H IY, A N G , B I H - Y I N G & C H E NC , H I - C H E N G1992. . Increase of xanthan production by cloning xps genes into wild-type Xanthomonas campestris. Letters in Applied Microbiology 1 4 , 4 3 4 6 Previously, genomic banks of Xanthomonas campestris were constructed in Escherichia coli, using mobilizable broad-host-range cosmids as the vectors. Following conjugal transfer, genes involved in the biosynthesis of xanthan polysaccharide (XPS) were cloned by the ability to restore the mucoid phenotype. to the non-mucoid mutants. In this study, all clones were transferred into the wild-type strain Xc17 to evaluate the effects of the cloned genes on XPS production. Most clones showed no significant effect; however, two plasmids, pP2401 and pP2201, caused 10 and 15% yield increases, respectively, compared with that of controls. While it was not clear how pP2201 caused the yield increase, the effect of pP2401 seemed to result from elevated phosphomannose isomerase activity. Since XPS synthesis in X. campestris is a very efficient process, only relatively small increases are to be expected; an enhancement of productivity by 1&15"/;1is important to the commercial production of xanthan Xanthan polysaccharide (XPS), produced by the phytopathogenic bacterium Xanthomonas campestris, is a heteropolymer which finds a variety of applications in agriculture and industry as a suspending, emulsifying and thickening agent (Kennedy & Bradshaw 1984). The chemical structure of X P S has been extensively studied and shown t o consist of a cellulosic (1 + 4)-b-Dglucose backbone with trisaccharide side chains attached t o alternate glucose residues. The trisaccharide side chain is composed of t w o mannose ( M a n ) units a n d one glucuronic acid (GluA) unit in the sequence of Man-GluA-Man (Jansson et a/. 1975; Melton et al. 1976; Lawson & Symes 1977) (Fig. 1). However, little is known about the genes. enzymes or mechanisms that control the synthesis of X P S by X . campestris. Recently, application of recombinant D N A techniques has resulted in several reports of the

* Corresponding author.

t

Present address: Center of Marine Biotechnology, Maryland Biotechnology Institute, University of Maryland, Baltimore, M D 21202, USA.

cloning of the genes involved in synthesis of XPS (Barrere et al. 1986; Harding et a f . 1987; Thorne et a/. 1987; H o t t e el a/. 1990). Efforts have also been made in our laboratory t o clone x p s genes. To achieve this end, two genomic banks of X . carnpestris were constructed in Escherichia coli using the RK2-derived, mobilizable cosmid p L A F R l (Friedman et a/. 1982) and pCP13 (Darzins & Chakrabarty 1984), respectively, as vectors. Following conjugal transfer of the banks en m u s e from E. coli into the non-mucoid mutants of X . campestris, wildtype genes were recovered as defined by their ability t o restore mucoid phenotype t o the

4 - 3 - I - G l t 1 - ( I--~)-B-D-GIU-(1-

I

P-D-hPan-( 1 -J)-,O-D-GluAi I -2)-a-D-Man-( 1-3) Fig. 1. Structure of pentasaccharide repeating unit of xanthan polysaccharide showing the trisaccharide side chain attached to the cellulosic (1 4 4)-B-D-glucose backbone (Glu, glucose; Man, mannose; GluA, glucuronic acid).

44

Yi-Hsiung Tseng et al.

mutant strains (Yang et al. 1987). A total of 18 clones with complementing ability were thus obtained. Mapping and hybridization data showed that some of these clones carried overlapping inserts. It was concluded that the xps genes reside on at least three regions in the X. carnpestris chromosome, with the longest one extending to 70 kb (Yang et al. 1990). All clones were transferred into the wild-type strain Xc17, and two were able to increase XPS production.

Materials and Methods CULTURE CONDITIONS

LB medium (Maniatis et al. 1982) was used as a general-purpose medium. XOLN (Fu & Tseng 1990), a liquid basal salt medium containing yeast extract (0.0625%) and tryptone (0.0625%), supplemented with glucose (1.2%), was used for measurement of XPS production. Liquid cultures of E. coli and X. campestris were grown at 37°C and 28"C, respectively, with vigorous shaking.

DNA TECHNIQUES

Plasmid DNA was prepared by the method of Birnboim & Doly (1979). Restriction endonucleases were purchased from Bethesda Research Laboratory and New England Biolabs, and used according to the instructions provided by the suppliers.

C O N J U G ATLR A N S F E R O F P L A S M I D S Recombinant plasmids were transferred from E . coli to Xc17 by triparental mating (Yang et al. 1988), with the help of pRK2013 (Ditta et al. 1980). Transconjugants were selected on LB agar containing ampicillin (Ap; 50 ,ug/ml) and tetracycline (Tc; 15 ,ug/ml), since Xc17 was ApR and the cells harbouring the recombinant plasmids were TcR.

D E T E R M I N A T I O N O F XPS C O N T E N T S

An overnight culture of an exconjugant was harvested by centrifugation and inoculated, at initial ODss0 of 0.35 (ca 1.75 x lo8 cells/ml),

into a 250 ml flask containing 40 ml of XOLN plus 1.2% glucose, then grown for 48 h. The cells were removed by centrifugation and the XPS in the supernatant was precipitated in the presence of 40 mM NaCl with 70% ethanol at - 20°C overnight. The pellet was washed with 70% ethanol and then resuspended in distilled water, The amount of XPS was measured by the anthrone method (Lin & Tseng 1979). Results and Discussion

Synthesis of XPS is a complex process involving multiple steps. If a rate-limiting enzymatic step exists in the biosynthetic pathway, then raising the copy number of the gene that confers the limiting enzyme would increase the XPS production. In this study, all previously obtained clones that possess the ability to complement the defect in XPS synthesis were each conjugally transferred from E. coli HBlOl into the wildtype strain Xc17. The exconjugants were then tested for XPS production in four independent tests with triplicate cultures. Most clones caused no significant increase in the productivity or the increase was not consistent. Two plasmids, pP2401 and pP2201, resulted in 10 and 15% yield increases of XPS, respectively, compared with that of controls without plasmid or with the vector alone (Table 1). Plasmids pP2201 and pP2401 carried EcoRI fragments of 4.1 and 3.8 kb, respectively, and possessed different restriction maps (Fig. 2). They restored the XPS synthesis ability to two distinctly different groups of non-mucoid mutants (Yang et al. 1990). DNA sequence analysis of the insert of pP2401 revealed two complete open reading frames (ORFs), while deletion mapping and enzyme assay identified the gene and function relationships between phosphomannose isomerase (PMI) and one of these ORF (Ting et al. 1990) (Fig. 2a). PMI is the first enzyme of the pathway converting

Table 1. Yield of xanthan polysaccharide (XPS) by Xanthomonas campestris Strain

XPS @g/ml)*

Yield(%)

Xc17 Xc17(pP2201) x c 17(pP2401)

6,363 7,296 7,012

100 115 110

*

Mean value of four independent experiments.

Increased xanthan production in X . campestris P I

E

P X P

S V P 1 1

I

K I

C

E

I

I

45

This study was supported by the National Science Council of the Republic of China.

References Fig. 2. Restriction maps of the inserts of (a) pP2401 (b) pP2201. Restriction sites for BamHI, BglII, ClaI, EcoRI, EcoRV, K p n l , Pstl, SmaI and XhoI are denoted by B, Bg. C , R, V. K, P, S and X, respectively.

fructose-6-phosphate via 6-phosphomannose and mannose- 1 -phosphate to GDP-mannose, the substrate for transferring the mannose moiety to form the side chains of XPS. Since the plasmid carrying deleted p m i gene failed to increase XPS production (data not shown), and the P M I activity in Xc17 (pP24011 was I.7-fold higher than that in the control Xc17 (Ting et al. 1990), it is likely that the effect on productivity increase was exerted by the increased P M I activity. In the pP2201 insert, two complete ORFs were also revealed; however, no enzyme function has been assigned to them yet (Chou 1991). The reasons for yield increase by this clone are therefore not clear Harding er a / . (1987) described a 10% increase of XPS yield. by cloning into wild-type cells clustered nornial u p s genes that resided on a D N A fragment of 12.4 kb. By the same strategy, Thorne er al. (1987) obtained three large cosmid clones that were able t o increase the XPS productikity up to 20%. Since several genes might be included in these clones, it was not determined which of the genes was ratelimiting. Furthermore, as insufficient information is available on the detailed physical maps and sequence homology with one another, it is not clear if the inserts of pP2201 and pP2401 are included in the previously reported clones (Harding er a!. 1985: Thorne et ul. 1987). Since synthesis o f XPS by X . campestris is a very efficient process (Moraine & Rogovin 1966), only relative11 small increases can be expected despite the fact that the introduced genes are multiplied (the copy number of p L A F R l and its isologous plasmids is about five in X . campesrris). Therefore, an enhancement of the productivity by 10-20u/u as shown in this study and by others (Harding et al. 1987; Thorne el a/., 1987) is significant for the commercial production of XPS.

BARREKE,G.C., BARBER.C.E. & DANIELS, M.J. 1986 Molecular cloning of genes involved in the production of the extracellular polysaccharide xanthan by Xanrhomonas compestris pv. mmpestris. International Journal of Biological Macromolecules 8, 312-374. BIRNBOIM, H.C. & DOLY.J. 1979 A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Research 7, 15131523.. CHOLJ,H.C. 1991 Cloning and sequence of a 4.1 kb EcoRI fragment that complements the non-mucoid mutant P?2 of Xanthomonas cumpestris pv. campestris. MS Thesis, National